This invention relates to apparatus and methods for coating specimens used in electron microscopy.
There are two techniques that are used for viewing specimens with an electron microscope. These two techniques are scanning electron microscopy (SEM) and transmitted electron microscopy (TEM).
Both SEM and TEM usually require that the specimen be coated. However, the required characteristics of the coating are different for the two viewing techniques.
For SEM the electron beam impacts the specimen and is rastered across the surface of the specimen to produce emitted secondary electrons, X-rays and other types of radiation from the coated surface. This secondary emitted radiation is then scanned and used to produce either an image on a cathode ray tube screen or on a photographic plate. The image appears as a three-dimensional image, much like the picture on a television set, and is a greatly magnified reproduction of the surface topography of the specimen.
Specimens prepared for viewing by SEM are often coated by a sputtering technique. This technique uses apparatus comprising a two-electrode glow discharge system having a cathode and anode. The cathode, which is made of the material to be deposited, is sputtered by means of an electrical glow discharge in an atmosphere of gas at a pressure generally between 1 to 10.sup.-2 torr. The sputtered cathode material is deposited on a substrate to form a film.
A sputtering apparatus made by Technics, Inc. and having the specimen mounted parallel to the cathode, is often used for coating specimens to be viewed by SEM.
In TEM the electron beam is transmitted through the specimen. The specimen in this case has to be electron dense to defract the electrons passing through the specimen. The different parts of the specimen defract the electron beam in different amounts, depending upon the composition and density of the different parts of the specimen. The defracted electrons are then used to produce the final image (which again can be displayed on a cathode ray tube or used to produce a negative for photographic print).
Coatings are used for TEM specimens to improve the electron beam defracting characteristics of the specimen and also to help stabilize the specimen as the electron beam is being transmitted through the specimen.
Specimens used for TEM are often coated by a film produced by evaporating electrodes of carbon or some other metal. An evaporative film coating apparatus made by Varian Associates, Inc. is often used for coating TEM specimens.
The prior art techniques for coating specimens for SEM have presented a number of problems.
All of the surface of the specimen to be viewed must be coated sufficiently so as to be conductive. Any part of the surface which is not sufficiently coated will charge, rather than emit the secondary radiation. This is a particular problem with biological specimens having undersurfaces and recessed surfaces that are difficult to present to the coating particles. Prior art coating apparatus which presented the specimen to the cathode in a planar or essentially a planar attitude often were not effective to provide the required coatings on all the surfaces of the specimen.
Overheating of the specimen during coating was another problem in the prior art, particularly in the case of biological specimens having fragile membranes which could be distorted or otherwise damaged by excessive heating. In attempts to avoid specimen overheating, the prior art often used water cooling on the base and also on the cathode. However, the prior art water cooling techniques still resulted in specimen overheating in some instances.
In TEM, overheating of the specimen by the transmitted electron beam (which is at about 110.degree. C) can be a problem. Overheating produces shrinking of the specimen and shifting and blurring of the image. This has been a definite problem with some specimens coated by prior art coating techniques. That is, the prior art coating techniques in many instances have not provided either the required accentuation of the varying density of the specimen or the required stabilizing of the specimen to permit long enough durations of exposure (without distortion of the specimen).